Chronic Graft versus Host disease (cGVHD) is a major limiting factor for the success of allo-HSCT. In this prospective study we aimed to evaluate the association between the kinetics of Regulatory T cells (TREG) and Conventional CD4+T cells (TCON) reconstitution and the emergence of cGVHD. We performed a detailed phenotypic analysis by multiparametric flow cytometry using fresh blood from 39 patients undergoing unrelated donor HSCT after a reduced intensity conditioning regimen containing ATG over a 2 year period, representing a total of 213 samples analyzed. GVHD prophylaxis consisted of cyclosporine plus mycophenolate mofetil. 11 patients were excluded due to disease relapse and/or death due to infection or acute GVHD in the first 9 months post-HSCT.We observed indiscriminately low numbers of TREG (CD4+CD127lowFoxp3+CD25bright) until mo 6 after HSCT and reduced TREG numbers in patients developing cGVHD (GVHD+) versus those who did not (GVHD-) (p=0.02 at mo 9).We further studied the dynamics of TREG subset reconstitution. CM TREG (CD45RA-CD62L+) was the predominant population in both patient groups. EM (CD45RA-CD62L-) was the second most abundant TREG subset. CM and EM TREG numbers were similar between patient groups. EMRA (CD45RA+CD62L-) TREG remained very low throughout the follow-up but were significantly increased at mo 9 in GVHD+ (p= 0.03). Interestingly, Naïve TREG (CD45RA+CD62L+CD95-) started to emerge at mo 9 and were significantly reduced in GVHD+ patients at mo 12 (0.71 vs 0.14 cells/µl; p=0.02)The Stem Cell Memory subset (SCM), identified as CD45RA+CD62L+CD95+, is thought to be self renewing and multipotent, being able to differentiate into CM, EM and TEMRA memory subsets. While in GVHD- SCM TREG started to emerge at mo 9, this subset remained low in GVHD+. Statistically significant differences were observed at mo 18 (0.91 vs 0.15 cells/µl; p=0.02).To ascertain the role of thymic output in TREG reconstitution we quantified CD31+ naïve TREG. Recent Thymic Emigrant cells (RTE) TREG were significantly reduced in GVHD+ at mo 12 (0.8 vs 0.16cells/µl; p=0.02) and at mo 18 (1.93 vs 0.28 cells/µl; p=0.02).In order to clarify whether both thymic output and peripheral expansion were contributing factors to decreased Naïve TREG, we quantified proliferation using Ki-67. TREG from GVHD+ patients proliferated less from months 2 to 18 reaching statistical significance at mo 9 and 12 (p= 0.003 and 0.02, respectively), suggesting that decreased TREG numbers are at least partly due to reduced peripheral expansion.Taken together, these observations suggest a compromised peripheral expansion and de novo generation of TREG through thymic output in cGVHD patients. Of note, susceptibility to apoptosis was not increased in TREG from GVHD+ patients, as Bcl-2 levels tended to be higher in relation to GVHD- patients.In the CD4+Foxp3- TCON population, we observed a clear predominance of EM in all patients. These cells, together with CM, are the first to appear at similar levels in both patient groups. EMRA emerged at higher numbers in GVHD+, although the results did not reach statistical significance.Naïve and RTE TCON started to emerge after mo 6 in GVHD-. Interestingly, these cells remained much lower in GVHD+ vs GVHD- patients throughout the follow-up, reaching statistical significance at mo 12 (p=0.03 for naïve; p=0.03 for RTE TCON).SCM TCON remained very low in all patients showing a tendency to be reduced in GVHD+ when compared to GVHD-. This difference reached statistical significance at mo 18 (6.85 vs 2.13 cells/µl; p=0.02).The small number of TREG and TCON subsets in GVHD+ patients were unlikely due to increased susceptibility to apoptosis, as assessed by Bcl-2 expression. The low numbers of cells within naïve and SCM TREG gates precluded the analysis of Bcl-2 expression in these subsets. Bcl-2 tended to be increased in total TREG CM and EM in GVHD+. In TCONs, Bcl-2 levels were similar in both patient groups.In summary, our data in patients developing cGVHD suggest that decreased thymic output and increased differentiation into terminally differentiated effector cells may have a negative impact on the number of naïve and SCM TCON and TREG. We speculate that the inability to generate and maintain the more immature TREG subsets may lead to decreased TREG numbers after 6 months post transplant, potentially resulting in decreased control of effector T cells contributing to the development of cGVHD. DisclosuresRitz:Kiadis: Membership on an entity's Board of Directors or advisory committees.